Stereoselective Synthesis of Bisfuranoxide (Aurochrome, Auroxanthin) and Monofuranoxide (Equinenone 5′,8′-Epoxide) Carotenoids by Double Horner–Wadsworth–Emmons Reaction

The stereoselective synthesis of C40-all-trans-carotenoids with the formal hexahydrobenzofuran skeletons aurochrome, auroxanthin, and equinenone-5′,8′-epoxide is reported. The synthesis is based on a one-pot or stepwise double Horner–Wadsworth–Emmons (HWE) reaction of a terminal enantiopure C15-5,6-epoxycyclohexadienylphosphonate and a central C10-trienedial. The ring expansion of the epoxycyclohexadienylphosphonate, generated by a Stille cross-coupling reaction, to the hexahydrobenzofuran skeleton was promoted by the reaction conditions of the HWE reaction prior to double-bond formation.

C arotenoids 1,2 are a group of numerous naturally occurring polyenic pigments ubiquitously present in the plant kingdom and other photosynthetic organisms, for which more than 750 compounds have been reported. 3,4 Being components of the photosynthetic 5 and photoprotective structural arrangements in these species, 6 carotenoids play fundamental roles in maintaining life. 7 Carotenoids are also responsible for the color and stability of some fruits, vegetables, flowers, and birds. 8 These polyenes hold potential as chemopreventive agents in humans by acting as antioxidants due to the radical-stabilizing ability of their conjugated unsaturated chains. 6,9 In addition, a plethora of bioactivities have been reported for carotenoids, 10 including anti-inflammatory, anticancer, 11 antimetabolic disorders, 12 and inhibition of lipid peroxidation. 13 Since the great majority of natural carotenoids are geometrically homogeneous all-trans polyene isomers, rather than differing in the doublebond geometries, they show large structural variability at the cyclohexenyl ring and also at the proximal double bonds. 14 Recent work in this field is focused on exploring the production of large quantities of these polyenic natural products by engineering a variety of carotenoid biosynthetic genes. 15 For example, the marine-bacterial carotenoid 4,4′ketolase (4,4′-oxygenase) gene crtW has been shown to promote the biogenesis of some 4-ketocarotenoids. Expression of the ketolase crtW gene in tubers of sweet potatoes [Iponomea batatas (L.) Lam] under the control of the CaMV promoter allowed the generation of novel carotenoids with furanoxide and cyclohexenone functional fragments, for example, echinenone 5′,8′-epoxide (2a and 2b, Scheme 1). 16 A 60:40 mixture of diastereomers of 2 was obtained when the sweet potato was extracted under normal conditions, 16 which suggested that these compounds might have been formed through the rearrangement of the putative precursor echinenone 5′,6′-epoxide (1, Scheme 1). 16 Echinenone-5′,8′-epoxides (2) belong to the small group of carotenoids termed epoxycarotenoids, which also include symmetrical members such as aurochrome and auroxanthin (4 and 6, respectively, Scheme 1), for which some promising biological activities have been reported. 17 The bis-furanoxide aurochrome (4, Scheme 1) has previously been isolated in very small amounts from green leaves of several Kenyan clones, 18,19 although its natural occurrence and biogenetic connection to β-carotene diepoxide (3, Scheme 1) have not been proven. Auroxanthin (6) has been isolated, together with its putative biogenetic precursor, the 5,6-epoxycarotenoid violaxanthin (5, Scheme 1), 3,4 from petals of the yellow Rosa fetidu HERRM, 20 eggs of hens seaweed meal, 21 and microalga Chlorella pyrenoidosa mutant G44. 22,23 From the former, auroxanthin (6) was isolated as a mixture of four diastereomers that differed by the configuration of the dihydrofuran ring (namely, 8R,8′S, 8S,8′S and 8R,8′R) and the geometry of one of the proximal double bonds (9′Z,8R,8′R). 20,24 The general assumption that the entire subfamily of carotenoids with furanoxide rings fused to the trimethylcyclohexane originates from the rearrangement of structurally related analogues containing 5′,6′-epoxide subunits upon exposure of these functionalities (Scheme 1) to acid media during the isolation and purification protocols 25 led to the consideration of these natural products as artifacts. 26−28 However, after careful control experiments, upon subjecting one of the putative butene monoepoxide precursors, i.e., peridinin (not shown), to these conditions, the furanoxide derivatives were not present in the reaction mixture. 29 Therefore, although peridinin also contains a γ-butenolide substructure, it is currently considered that the furanoxides might indeed be true natural products and not artifacts.

■ RESULTS AND DISCUSSION
Following the bidirectional approach to carotenoid synthesis, the doubly functionalized iodo-butenylphosphonate 12 was needed to accomplish the orthogonal Stille cross-coupling and HWE reaction steps of the planned synthesis. This fragment was prepared in 60% overall yield as previously described (Scheme 3). 31 On route to aurochrome (4), the palladium-catalyzed Stille cross-coupling reaction promoted by the cocatalytic effect of Cu(I) 46 of alkenyl iodide 12 and alkenylstannane 13 47 afforded epoxypentadienylphosphonate 8 in almost quantitative yield. Reaction conditions for carotenoid formation were first explored by following the procedure described for the racemic material. 28,45 Upon treatment of 8 with KOtBu (tetrahydrofur-an (THF), −30°C) and reaction of the anion with 7, the predominant formation of the thermodynamically favored 48 all-trans isomer of the polyene skeleton of aurochrome (4) was generated as a 3:1 mixture of diastereomers (Scheme 3A). 45 A complex reaction mechanism has already been proposed under the reaction conditions to provide aurochrome (4, Scheme 3A) following formation of the phosphonate anion I stabilized through conjugation, namely, (i) ring-opening of the 5,6epoxide; (ii) ring-closure by conjugate addition of the generated alkoxide to the trienylphosphonate intermediate II (Scheme 3B) to afford the reacting alkenyl-5,8-epoxide phosphonate anion III; 45,49 and (iii) the 2-fold condensation with C 10 -trienedial 7. 28,45 Under basic conditions, the I to III rearrangement was expected to lead predominantly to the formation of the most stable furanoxide phosphonate anion isomer (III) and, therefore, to the all-trans isomer of the major diastereomer (53% yield), namely, (5R,8R,5′R,8′R)-aurochrome (4) (Scheme 3). 49 1 H NMR data confirmed this assumption, since it has been shown that Δδ H7−H8 for this diastereomer is very small (0.02 ppm) and the signal for H7 appears as a broad singlet. 27,28 The 8R/8′R configuration of the newly formed C8 and C8′ stereocenters for the major diastereomer was further confirmed by the NOE effect observed between the methyl groups at C5/C5′ and proton signals at C8/C8′. A similar approach was followed for the synthesis of the carotenoid 5,8-furanoxide auroxanthin (6). Combination of alkenyl iodide 12 and the previously described alkenylstannane 14 49,50 also using the Stille reaction performed under Furstner's conditions 46 provided the epoxydienyl phosphonate 9 (Scheme 3). With fragments (C 15 and C 10 ) in hand, the stereoselective synthesis of auroxanthin (6) followed the same procedure described for aurochrome (4). Thus, the reaction of 7 with the anion generated upon treatment of phosphonate-5,6-epoxide 9 with an excess of base (4.6 equiv of KOtBu) in THF afforded, after allowing the mixture to react from −30 to 0°C, auroxanthin (6) in 65% yield in a 3:1 diastereoisomeric ratio (Scheme 3). As indicated for the synthesis of aurochrome (4, Scheme 3B), the conjugate addition of the alkoxide anion to C8 of the trienyl phosphonate intermediate should afford the corresponding 5,8-furanoxide allyl phosphonate, itself involved as intermediate in the HWE condensation with triene dialdehyde 7 to provide auroxanthin (6).
Alternative protocols were explored for the synthesis of enantiopure auroxanthin (6), by changing the reaction components and by using phosphonate 10 (Scheme 2) and also acid-promoted rearrangement of the epoxydiene fragment 9. However, the outcome of these alternative procedures was disappointing (see the SI for a detailed study).

■ CONCLUSIONS
In summary, the HWE condensation reaction using a synthetic scheme based on a C 15 + C 10 + C 15 = C 40 pattern has been demonstrated to be a powerful tool for the stereocontrolled synthesis of the major 5R,8R diastereomers of enantiopure aurochrome (4), auroxanthin (6), and recently reported echinenone 5′,8′-epoxide (2a). This strategy makes use of a central C 10 -dialdehyde 7 and terminal enantiopure C 15dienylphosphonates having a C5,C6-epoxide as a common functionality. An HWE reaction and stereoretentive C5,C6 epoxide ring expansion to the C5,C8 dihydrofuran catalyzed by the basic media concomitantly took place and provided the 5,8-dihydrofuranoxide skeletons of aurochrome (4) and auroxanthin (6) in a 3:1 diastereomeric ratio. Moreover, for echinenone 5′,8′-epoxide (2a/b) the process was performed in a stepwise manner, with the second HWE reaction conditions promoting both C5,C6 epoxide ring expansion to C5,C8 dihydrofuran and double-bond formation and affording the nonsymmetrical carotenoid in a 4:1 diastereomeric ratio. The low efficiency of the putative biogenesis of echinenone 5′,8′epoxide (2a/2b) from crtW gene-expressed sweet potato tubers (0.1 mg was isolated from 100 g), which prevented the separation of these diastereomers, 16 further supports the relevance of the chemical synthesis to access the required amounts of these substrates for further analysis and biological evaluation.

■ EXPERIMENTAL SECTION
General Experimental Procedures. Solvents were dried according to published methods and distilled before use except THF, CH 2 Cl 2 , CH 3 CN, MeOH, Et 2 O, and DMF, which were dried using a Puresolv solvent purification system. All other reagents were commercial compounds of the highest purity available. All reactions were carried out under an argon atmosphere, and those not involving aqueous reagents were carried out in oven-dried glassware. All solvents and anhydrous solutions were transferred through syringes and cannulae, previously dried in the oven for at least 12 h and kept in a desiccator with KOH. Et 3 N, acetone, diisopropylamine, N,Ndiisopropylethylamine (DIPEA), and pyridine were dried by distillation from CaH 2 . Distillations were carried out in a Buchi GKR-50 Kugelrohr, and in that case the boiling points indicate the external temperature. For fractional distillations a microstill was used with an internal thermometer in the distillation head. The nBuLi concentration was determined by titration in triplicate with diphenyl acetic acid or N-pivaloyl-o-toluidine in THF at 0°C. For reactions at low temperature, ice/water or CO 2 /acetone systems were used. Alternatively, an AnaLogix Intelliflash 310 HPFC flash collector system was used. IR spectra were obtained with a JASCO FTIR 4200 spectrophotometer, from a thin film deposited onto NaCl glass. Specific rotations were measured on a JASCO P-1020 polarimeter with a Na lamp. HPLC was performed using a Waters instrument by using a dual-wavelength detector and a 3.5 × 100 mm glass cell. UV were developed in a Cary C BIO spectrometer in methanol as solvent. HRMS (ESI + ) were measured with an Apex III FTICR mass spectrometer (Bruker Daltonics). 1 H NMR spectra and 13 C NMR spectra were recorded in CDCl 3 , C 6 D 6 , CD 3 OD, and (CD 3 ) 2 CO at ambient temperature on a Bruker AMX-400 spectrometer operating at 400. 16 and 100.62 MHz with residual protic solvent as the internal reference (CDCl 3 , δ = 7.26 ppm; C 6 D 6 , δ = 7.16 ppm; (CD 3 ) 2 CO, δ = 2.05 ppm; and CD 3 OD, δ = 4.87 ppm) for the former and CDCl 3 (δ C = 77.2 ppm), C 6 D 6 (δ C = 128.0 ppm), (CD 3 ) 2 CO (δ C = 29.8 ppm), and CD 3 OD (δ C = 49.0 ppm) as the internal reference for the latter. Chemical shifts (δ) are given in parts per million (ppm), and coupling constants (J) are given in hertz (Hz). The proton spectra are reported as follows: δ (multiplicity, coupling constant J, number of protons). A DEPT-135 pulse sequence was used to aid in the assignment of signals in the 13 C NMR spectra. Multiplicity in the 13 C NMR spectral data refers to the attached hydrogens.